Multiscale morphogenesis of the mouse blastocyst by actomyosin contractility

During preimplantation development, the mouse embryo forms the blastocyst, which consists of a squamous epithelium enveloping a fluid-filled lumen and a cluster of pluripotent cells. The shaping of the blastocyst into its specific architecture is a prerequisite to implantation and further development of the embryo. Recent studies identified the central role of the actomyosin cortex in generating the forces driving the successive steps of blastocyst morphogenesis. As seen in other developing animals, actomyosin functions across spatial scales from the subcellular to the tissue levels. In addition, the slow development of the mouse embryo reveals that actomyosin contractility operates at multiple timescales with periodic cortical waves of contraction every ∼80 s and tissue remodeling over hours.     

Changes in the distribution of a spectrin-like protein during development of the preimplantation mouse embryo

The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte alpha-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.     

Asymmetric distribution of PAR proteins in the mouse embryo begins at the 8-cell stage during compaction

In many organisms, like Caenorhabditis elegans and Drosophila melanogaster, establishment of spatial patterns and definition of cell fate are driven by the segregation of determinants in response to spatial cues, as early as oogenesis or fertilization. In these organisms, a family of conserved proteins, the PAR proteins, is involved in the asymmetric distribution of cytoplasmic determinants and in the control of asymmetric divisions. In the mouse embryo, it is only at the 8-cell stage during compaction that asymmetries, leading to cellular diversification and blastocyst morphogenesis, are first observed. However, it has been suggested that developmentally relevant asymmetries could be established already in the oocyte and during fertilization. This led us to study the PAR proteins during the early stages of mouse development. We observed that the homologues of the different members of the PAR/aPKC complex and PAR1 are expressed in the preimplantation mouse embryo. During the first embryonic cleavages, before compaction, PARD6b and EMK1 are observed on the spindle. The localization of these two proteins becomes asymmetric during compaction, when blastomeres flatten upon each other and polarize. PARD6b is targeted to the apical pole, whereas EMK1 is distributed along the baso-lateral domain. The targeting of EMK1 is dependent upon cell-cell interactions while the apical localization of PARD6b is independent of cell contacts. At the 16-cell stage, aPKCzeta colocalizes with PARD6b and a colocalization of the three proteins (PARD6b/PARD3/aPKCzeta can occur in blastocysts, only at tight junctions. This choreography suggests that proteins of the PAR family are involved in the setting up of blastomere polarity and blastocyst morphogenesis in the early mammalian embryo although the interactions between the different players differ from previously studied systems. Finally, they reinforce the idea that the first developmentally relevant asymmetries are set up during compaction.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.     

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20-30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic-abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.     

The maternal‐to‐zygotic transition: An asynchronous split

The early cell cycles of preimplantation embryo development are unique in the scheme of mitotic cell proliferation as cell division is not coupled to cell growth, leading to a halving of blastomere volume with each cleavage event. Among the early mouse embryonic divisions, the fi rst two are particularly different, lasting almost twice as long as subsequent divisions. The third cell cycle is marked by the transition of a four‐cell embryo into an eight‐cell embryo, and represents the fi rst complete cell cycle occurring after activation of the zygotic genome. The G2/M phase of the third cell cycle is highly variable, lasting between 2–5 hours, and heterogeneity between blastomeres within the same embryo may occur as a part of normal development. The embryo in this image is actively undergoing cleavage from the four‐ to the eight‐cell stage, and blastomeres are captured in multiple phases of the cell cycle, as visualized by chromatin structure (DNA, blue) and microtubule staining (α‐tubulin, green). Two blastomeres sit in interphase with decondensed chromatin masses and a mesh‐like microtubule network, while the remaining blastomeres are actively undergoing mitosis. Of the latter, one is in metaphase, one in early anaphase, and the last in late anaphase. All together, the diversity in cell cycle stages reveals the inherit asynchrony existent within individual blastomeres of a cleavage stage embryo. Mol. Reprod. Dev. 80: 1–1, 2012. © 2012 Wiley Periodicals, Inc.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Tropomyosin in Preimplantation Mouse Development: Identification, Expression, and Organization during Cell Division and Polarization

Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS–PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higherM_risoforms were not represented, also some of the isoforms can be labeled by incorporation of [¹⁴C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.     

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Inactivation of aPKCλ Reveals a Context Dependent Allocation of Cell Lineages in Preimplantation Mouse Embryos

Background

During mammalian preimplantation development, lineage divergence seems to be controlled by the interplay between asymmetric cell division (once cells are polarized) and positional information. In the mouse embryo, two distinct cell populations are first observed at the 16-cell stage and can be distinguished by both their position (outside or inside) and their phenotype (polarized or non-polarized). Many efforts have been made during the last decade to characterize the molecular mechanisms driving lineage divergence.

Methodology/Principal Findings

In order to evaluate the importance of cell polarity in the determination of cell fate we have disturbed the activity of the apical complex aPKC/PAR6 using siRNA to down-regulate aPKCλ expression. Here we show that depletion of aPKCλ results in an absence of tight junctions and in severe polarity defects at the 16-cell stage. Importantly, we found that, in absence of aPKCλ, cell fate depends on the cellular context: depletion of aPKCλ in all cells results in a strong reduction of inner cells at the 16-cell stage, while inhibition of aPKCλ in only half of the embryo biases the progeny of aPKCλ defective blastomeres towards the inner cell mass. Finally, our study points to a role of cell shape in controlling cell position and thus lineage allocation.

Conclusion

Our data show that aPKCλ is dispensable for the establishment of polarity at the 8-cell stage but is essential for the stabilization of cell polarity at the 16-cell stage and for cell positioning. Moreover, this study reveals that in addition to positional information and asymmetric cell divisions, cell shape plays an important role for the control of lineage divergence during mouse preimplantation development. Cell shape is able to influence both the type of division (symmetric or asymmetric) and the position of the blastomeres within the embryo.     

Chromatin condensation activity and cortical activity during the first three cell cycles of a mouse embryo

One-cell parthenogenetic haploid embryos and blastomeres of the 2- and 4-cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one-cell embryos and of blastomeres. The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2- and 4-cell embryos did not exhibit any cortical activity. In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2-cell and 4-cell stage blastomeres did not reveal condensation activity. © 1995 Wiley-Liss, Inc.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos

The peripheral region of ascidian oocytes and zygotes contains five determinants for morphogenesis and differentiation of the embryo. The determinant for the 24 primary muscle cells of the tadpole, macho1, is one of several cortical mRNAs localized in a gradient along the animal-vegetal axis in the oocyte. After fertilization these mRNAs, together with cortical endoplasmic reticulum (cER) and a subcortical mitochondria-rich domain (myoplasm), relocate in two major reorganization phases forming the posterior plasm (postplasm) of the zygote. At the 8-cell stage cortical mRNAs concentrate in a macroscopic cortical structure called the centrosome-attracting body (CAB), forming a characteristic posterior end mark (PEM) in the two posterior vegetal blastomeres. We propose to call the numerous mRNAs showing this particular cortical localization in the posterior region of the embryo postplasmic/PEM RNAs and suggest a nomemclature. We do not know how postplasmic/PEM RNAs reach their polarized distribution in the oocyte cortex but at least PEM1 and macho1 (and probably others) bind to the network of cER retained in isolated cortical fragments. We propose that after fertilization, these postplasmic/PEM mRNAs move in the zygote cortex together with the cER network (cER/mRNA domain) via microfilament- and microtubule-driven translocations. The cER/mRNA domain is localized posteriorly at the time of first cleavage and distributed equally between the first two blastomeres. After the third cleavage, the cER/mRNA domain and dense particles compact to form the CAB in posterior vegetal blastomeres of the 8-cell stage. We discuss the identity of postplasmic/PEM RNAs, how they localize, anchor, relocate and may be translated. We also examine their roles in unequal cleavage and as a source of posterior morphogenetic and differentiation factors.     

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.